Cold-adapted equine influenza viruses

ABSTRACT

The present invention provides experimentally-generated cold-adapted equine influenza viruses, and reassortant influenza A viruses comprising at least one genome segment of such an equine influenza virus, wherein the equine influenza virus genome segment confers at least one identifying phenotype of the cold-adapted equine influenza virus, such as cold-adaptation, temperature sensitivity, dominant interference, or attenuation. Such viruses are formulated into therapeutic compositions to protect animals from diseases caused by influenza A viruses, and in particular, to protect horses from disease caused by equine influenza virus. The present invention also includes methods to protect animals from diseases caused by influenza A virus utilizing the claimed therapeutic compositions. Such methods include using a therapeutic composition as a vaccine to generate a protective immune response in an animal prior to exposure to a virulent virus, and using a therapeutic composition as a treatment for an animal that has been recently infected with a virulent virus, or is likely to be subsequently exposed to virulent viruses in a few days whereby the therapeutic composition interferes with the growth of the virulent virus, even in the absence of immunity. The present invention also provides methods to produce cold-adapted equine influenza viruses, and reassortant influenza A viruses having at least one genome segment of an equine influenza virus generated by cold-adaptation.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a 35 U.S.C. 371 of International PCT Application No.PCT/US01/05048, filed Feb. 16, 2001; which is a continuation-in-part ofU.S. patent application Ser. No. 09/506,286, filed Feb. 16, 2000, nowissued as U.S. Pat. No. 6,482,414 B1; all entitled “COLD-ADAPTED EQUINEINFLUENZA VIRUSES” and incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to nucleic acids and proteins ofexperimentally-generated cold-adapted equine influenza viruses, andparticularly to cold-adapted equine influenza viruses having additionalphenotypes, such as attenuation, dominant interference, or temperaturesensitivity. The invention also includes reassortant influenza A viruseswhich contain at least one genome segment from such an equine influenzavirus, such that the reassortant virus includes certain phenotypes ofthe donor equine influenza virus. The invention further includesgenetically-engineered equine influenza viruses, produced throughreverse genetics, which comprise certain identifying phenotypes of acold-adapted equine influenza virus of the present invention. Thepresent invention also relates to the use of these viruses intherapeutic compositions to protect animals from diseases caused byinfluenza viruses.

BACKGROUND OF THE INVENTION

Equine influenza virus has been recognized as a major respiratorypathogen in horses since about 1956. Disease symptoms caused by equineinfluenza virus can be severe, and are often followed by secondarybacterial infections. Two subtypes of equine influenza virus arerecognized, namely subtype-1, the prototype being A/Equine/Prague/1/56(H7N7), and subtype-2, the prototype being A/Equine/Miami/1/63 (H3N8).Presently, the predominant virus subtype is subtype-2, which has furtherdiverged among Eurasian and North American isolates in recent years. Thecurrently licensed vaccine for equine influenza is an inactivated(killed) virus vaccine. This vaccine provides minimal, if any,protection for horses, and can produce undesirable side effects, forexample, inflammatory reactions at the site of injection. See, e.g.,Mumford, 1987, Equine Infectious Disease IV, 207–217, and Mumford, etal., 1993, Vaccine 11, 1172–1174. Furthermore, current modalities cannotbe used in young foals, because they cannot overcome maternal immunity,and can induce tolerance in a younger animal. Based on the severity ofdisease, there remains a need for safe, effective therapeuticcompositions to protect horses against equine influenza disease.

Production of therapeutic compositions comprising cold-adapted humaninfluenza viruses is described, for example, in Maassab, et al., 1960,Nature 7,612–614, and Maassab, et al., 1969, J. Immunol. 102, 728–732.Furthermore, these researchers noted that cold-adapted human influenzaviruses, i.e., viruses that have been adapted to grow at lower thannormal temperatures, tend to have a phenotype wherein the virus istemperature sensitive; that is, the virus does not grow well at certainhigher, non-permissive temperatures at which the wild-type virus willgrow and replicate. Various cold-adapted human influenza A viruses,produced by reassortment with existing cold-adapted human influenza Aviruses, have been shown to elicit good immune responses in vaccinatedindividuals, and certain live attenuated cold-adapted reassortant humaninfluenza A viruses have proven to protect humans against challenge withwild-type virus. See, e.g., Clements, et al., 1986, J. Clin. Microbiol.23, 73–76. In U.S. Pat. No. 5,149,531, by Youngner, et al., issued Sep.22, 1992, the inventors of the present invention further demonstratedthat certain reassortant cold-adapted human influenza A viruses alsopossess a dominant interference phenotype, i.e., they inhibit the growthof their corresponding parental wild-type strain, as well asheterologous influenza A viruses.

U.S. Pat. No. 4,683,137, by Coggins et al., issued Jul. 28, 1987, andU.S. Pat. No. 4,693,893, by Campbell, issued Sep. 15, 1987, discloseattenuated therapeutic compositions produced by reassortment ofwild-type equine influenza viruses with attenuated, cold-adapted humaninfluenza A viruses. Although these therapeutic compositions appear tobe generally safe and effective in horses, they pose a significantdanger of introducing into the environment a virus containing both humanand equine influenza genes.

SUMMARY OF THE INVENTION

The present invention provides nucleic acids and proteins ofexperimentally-generated cold-adapted equine influenza viruses, andreassortant influenza A viruses.

Examples of cold-adapted equine influenza viruses of the presentinvention include EIV-P821, identified by accession No. ATCC VR-2625;EIV-P824, identified by accession No. ATCC VR-2624; EIV-MSV+5,identified by accession No. ATCC VR-627; and progeny of such viruses.Cold-adapted equine influenza viruses of the invention, and theirmethods of making, are disclosed in related U.S. Pat. No. 6,177,082, byDowling et al., issued Jan. 23, 2001; and WO 00/09702, by Dowling etal., published Feb. 24, 2000, both of which are incorporated herein byreference in their entirety.

The present invention also describes nucleic acid molecules encodingwild-type and cold-adapted equine influenza proteins PB2, NS, PB1, PAand NA. One embodiment of the present invention is an isolated equinenucleic acid molecule having a nucleic acid sequence selected from agroup consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:13, SEQ ID NO:19, SEQ ED NO:20, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39,SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47,SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:60,SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:68,SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:82,SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:89,SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:100and SEQ ID NO:102 and a nucleic acid molecule comprising a nucleic acidsequence which is fully complementary to any of such nucleic acidsequences. Another embodiment of the present invention is an isolatedequine nucleic acid molecule that encodes a protein comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:6, SEQ IDNO:9, SEQ ID NO:14, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ IDNO:33, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ IDNO:49, SEQ ID NO:54, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:85, SEQ ID NO:88, SEQ IDNO:96, SEQ ID NO:99, SEQ ID NO:101 and SEQ ID NO:103. Another embodimentis an isolated equine influenza protein that comprises an amino acidsequence selected from a group consisting of SEQ ID NO:6, SEQ ID NO:9,SEQ ID NO:14, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:33,SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49,SEQ ID NO:54, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:69,SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:96,SEQ ID NO:99, SEQ ID NO:101 and SEQ ID NO:103. Also included in thepresent invention is a virus that include any of these nucleic acidmolecules or proteins. In one embodiment, such a virus is equineinfluenza virus or a reassortant virus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides nucleic acids and proteins ofexperimentally-generated cold-adapted equine influenza virusescomprising certain defined phenotypes, which are disclosed herein. It isto be noted that the term “a” or “an” entity, refers to one or more ofthat entity; for example, “a cold-adapted equine influenza virus” caninclude one or more cold-adapted equine influenza viruses. As such, theterms “a” (or “an”), “one or more,” and “at least one” can be usedinterchangeably herein. It is also to be noted that the terms“comprising,” “including,” and “having” can be used interchangeably.Furthermore, an item “selected from the group consisting of” refers toone or more of the items in that group, including combinations thereof.

A cold-adapted equine influenza virus of the present invention is avirus that has been generated in the laboratory, and as such, is not avirus as occurs in nature. Since the present invention also includesthose viruses having the identifying phenotypes of such a cold-adaptedequine influenza virus, an equine influenza virus isolated from amixture of naturally-occurring viruses, i.e., removed from its naturalmilieu, but having the claimed phenotypes, is included in the presentinvention. A cold-adapted equine influenza virus of the presentinvention does not require any specific level of purity. For example, acold-adapted equine influenza virus grown in embryonated chicken eggsmay be in a mixture with the allantoic fluid (AF), and a cold-adaptedequine influenza virus grown in tissue culture cells may be in a mixturewith disrupted cells and tissue culture medium.

As used herein, an “equine influenza virus” is an influenza virus thatinfects and grows in equids, e.g., horses or ponies. As used herein,“growth” of a virus denotes the ability of the virus to reproduce or“replicate” itself in a permissive host cell. As such, the terms,“growth of a virus” and “replication of a virus” are usedinterchangeably herein. Growth or replication of a virus in a particularhost cell can be demonstrated and measured by standard methodswell-known to those skilled in the art of virology. For example, samplescontaining infectious virus, e.g., as contained in nasopharyngealsecretions from an infected horse, are tested for their ability to causecytopathic effect (CPE), e.g., virus plaques, in tissue culture cells.Infectious virus may also be detected by inoculation of a sample intothe allantoic cavity of embryonated chicken eggs, and then testing theAF of eggs thus inoculated for its ability to agglutinate red bloodcells, i.e., cause hemagglutination, due to the presence of theinfluenza virus hemagglutinin (HA) protein in the AF.

Cold-adapted equine influenza viruses of the present invention arecharacterized primarily by one or more of the following identifyingphenotypes: cold-adaptation, temperature sensitivity, dominantinterference, and/or attenuation. As used herein, the phrase “an equineinfluenza virus comprises the identifying phenotype(s) ofcold-adaptation, temperature sensitivity, dominant interference, and/orattenuation” refers to a virus having such a phenotype(s). Examples ofsuch viruses include, but are not limited to, EIV-P821, identified byaccession No. ATCC VR-2625, EIV-P824, identified by accession No. ATCCVR-2624, and EIV-MSV+5, identified by accession No. ATCC VR-2627, aswell as EIV-MSV0, EIV, MSV+1, EIV-MSV+2, EIV-MSV+3, and EIV-MSV+4.

Pursuant to 37 CFR § 1.802 (a–c), cold-adapted equine influenza viruses,designated herein as EIV-P821, an EIV-P824 were deposited with theAmerican Type Culture Collection (ATCC, 10801 University Boulevard,Manassas, Va. 20110-2209) under the Budapest Treaty as ATCC AccessionNos. ATCC VR-2625, and ATCC VR-2624, respectively, on Jul. 11, 1998.Cold-adapted equine influenza virus EIV-MSV+5 was deposited with theATCC as ATCC Accession No. ATCC VR-2627 on Aug. 3, 1998. Pursuant to 37CFR§ 1.806, the deposits are made for a term of at least thirty (30)years and at least five (5) years after the most recent request for thefurnishing of a sample of the deposit was received by the depository.Pursuant to 37 CFR § 1.808 (a)(2), all restrictions imposed by thedepositor on the availability to the public will be irrevocably removedupon the granting of the patent.

The present invention includes nucleic acid molecules isolated fromequine influenza virus wild type strain A/equine/Kentucky/1/91 (H3N8),and cold-adapted equine influenza virus EIV-P821.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subject to human manipulation) andcan include DNA, RNA, or derivatives of either DNA or RNA. As such,“isolated” does not reflect the extent to which the nucleic acidmolecule has been purified.

The present invention includes nucleic acid molecules encoding wild-typeand cold-adapted equine influenza virus proteins. Nucleic acid moleculesof the present invention can be prepared by methods known to one skilledin the art. Proteins of the present invention can be prepared by methodsknown to one skilled in the art, i.e., recombinant DNA technology.Preferred nucleic acid molecules have coding strands comprising nucleicacid sequences SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:13, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:60, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:82, SEQ IDNO:83, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:94, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:100 and SEQID NO:102 and/or a complement thereof. Complements are defined as twosingle strands of nucleic acid in which the nucleotide sequence is suchthat they will hybridize as a result of base pairing throughout theirfull length. Given a nucleotide sequence, one of ordinary skill in theart can deduce the complement.

Preferred nucleic acid molecules encoding equine influenza PB2 proteinsare nei_(wt)PB2₂₃₄₁, nei_(wt)PB2₂₂₇₇, nei_(ca1)PB2₂₃₄₁, and/ornei_(ca1)PB2₂₂₇₇, the coding strands of which are represented by SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:27, and/or SEQ ID NO:29.

Preferred nucleic acid molecules encoding equine influenza NS proteinsare nei_(wt1)NS₈₉₁, nei_(wt1)NS₆₉₀, nei_(wt3)NS₈₈₈, nei_(wt4)NS₄₆₈,nei_(wt4)NS₂₉₃, nei_(ca1)NS₈₈₈, and/or nei_(ca1)NS₆₉₀, the codingstrands of which are represented by SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39 and/or SEQ ID NO:41.

Preferred nucleic acid molecules encoding equine influenza PB1-Nproteins are nei_(wt1)PB1-N₁₂₂₉, nei_(wt1)PB1-N₁₁₉₄, nei_(wt2)PB1-N₆₇₃,nei_(wt2)PB1-N₆₃₆, nei_(ca1)PB1-N₁₂₂₅, nei_(ca1)PB1-N₁₁₈₅,nei_(ca2)PB1-N₁₂₂₁, and/or nei_(ca2)PB1-N₁₁₈₅, the coding strands ofwhich are represented by SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQID NO:47, SEQ ID NO:48, SEQ ID NO:50, and/or SEQ ID NO:51.

Preferred nucleic acid molecules encoding equine influenza PB1-Cproteins are nei_(wt1)PB1-C₁₂₃₄, nei_(wt1)PB1-C₁₁₈₈, nei_(wt2)PB1-C₁₂₄₀,nei_(ca1)PB1-C₁₂₄₁, nei_(ca1)PB1-C₁₁₈₈, and/or nei_(ca2)PB1-C₁₂₄₁, thecoding strands of which are represented by SEQ ID NO:53, SEQ ID NO:60,SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:66, and/or SEQ IDNO:94.

Preferred nucleic acid molecules encoding equine influenza PB1 proteinsare nei_(wt)PB1₂₃₄₁, nei_(wt)PB1₂₂₇₁, nei_(ca1)PB1₂₃₄₁,nei_(ca1)PB1₂₂₇₁, the coding strands of which are represented by SEQ IDNO:68, SEQ ID NO:70, SEQ ID NO:71, and/or SEQ ID NO:73.

Preferred nucleic acid molecules encoding equine influenza PA-C proteinsare nei_(wt1)PA-C₁₂₂₈, nei_(wt1)PA-C₁₁₆₄, nei_(wt2)PA-C₁₂₂₃,nei_(ca1)PA-C₁₂₃₃, and nei_(ca1)PA-C₁₁₇₀, the coding strands of whichare represented by SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:83, SEQ IDNO:84, and/or SEQ ID NO:86.

Preferred nucleic acid molecules encoding equine influenza PA-N proteinsare nei_(wt)PA-N₁₂₁₆, nei_(wt)PA-N₁₁₉₃, nei_(ca)PA-N₁₂₁₇, andnei_(ca)PA-N₁₁₉₃, the coding strands of which are represented by SEQ IDNO:94, SEQ ID NO:95, SEQ ID NO:97 and SEQ ID NO:98.

Preferred nucleic acid molecules encoding equine influenza PA proteinsare nei_(wt)PA₂₁₄₈ and nei_(ca)PA₂₁₄₈, the coding strands of which arerepresented by SEQ ID NO:100 and SEQ ID NO:102.

Preferred nucleic acid molecules encoding equine influenza NA proteinsare nei_(ca)NA₁₄₇₈ and nei_(ca)NA₁₄₁₀, the coding strands of which arerepresented by SEQ ID NO:87 and SEQ ID NO:89.

The present invention includes proteins comprising SEQ ID NO:6, SEQ IDNO:9, SEQ ID NO:14, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ IDNO:33, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ IDNO:49, SEQ ID NO:54, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:67, SEQ IDNO:69, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:85, SEQ ID NO:88, SEQ IDNO:96, SEQ ID NO:99, SEQ ID NO:101 and SEQ ID NO:103 as well as nucleicacid molecules encoding such proteins.

Preferred equine influenza PB2-N proteins of the present inventioninclude proteins encoded by a nucleic acid molecule comprisingnei_(wt)PB2-N₁₂₄₁, nei_(wt)PB2-N₁₂₁₄, nei_(ca1)PB2-N₁₂₄₁,nei_(ca1)PB2-N₁₂₁₄ nei_(ca2), and/or PB2-N₁₂₁₄. Preferred equineinfluenza PB2-N proteins are P_(wt)PB2-N₄₀₄, P_(ca1)PB2-N₄₀₄, and/orP_(ca2)PB2-N₄₀₄. In one embodiment, a preferred equine influenza PB2-Nprotein of the present invention is encoded by SEQ ID NO:5, SEQ IDNO:7,SEQ ID NO:8, and/or SEQ ID NO:10, and, as such, has an amino acidsequence that includes SEQ ID NO:6 and/or SEQ ID NO:9.

Preferred equine influenza PB2-C proteins of the present inventioninclude proteins encoded by a nucleic acid molecule comprisingnei_(wt1)PB2C-₁₂₃₃, nei_(wt2)PB2-C₁₂₃₂, nei_(wt)PB2-C₁₁₉₄,nei_(ca1)PB2-C₁₂₃₂, and/or nei_(ca1)PB2-C₁₁₉₄. In one embodiment, apreferred equine influenza PB2-C protein of the present invention isencoded by SEQ ID NO:13, SEQ ID NO:20, SEQ ID NO:19, SEQ ID NO:21,and/or SEQ ID NO:23, and, as such, has an amino acid sequence thatincludes SEQ ID NO:14 and/or SEQ ID NO:22.

Preferred equine influenza PB2 proteins of the present invention includeproteins encoded by a nucleic acid molecule comprising nei_(wt)PB2₂₃₄₁,nei_(wt)PB2₂₂₇₇, nei_(ca1)PB2₂₃₄₁, and or nei_(ca1)PB2₂₂₇₇. In oneembodiment, a preferred equine influenza PB2 protein of the presentinvention is encoded by SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, and/orSEQ ID NO:29, and, as such, has an amino acid sequence that includes SEQID NO:25 and/or SEQ ID NO:28.

Preferred equine influenza NS proteins of the present invention includeproteins encoded by a nucleic acid molecule comprising nei_(wt1)NS₈₉₁,nei_(wt1)NS₆₉₀, nei_(wt3)NS₈₈₈, nei_(wt4)NS₄₆₈, nei_(wt4)NS₂₉₃,nei_(ca1)NS₈₈₈, and/or nei_(ca1)NS₆₉₀. Preferred equine influenza NSproteins are Pei_(wt)NS₂₃₀, Pei_(wt4)NS₉₇, and/or Pei_(ca1)NS₂₃₀. In oneembodiment, a preferred equine influenza NS protein of the presentinvention is encoded by SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:35, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:39 and/or SEQ ID NO:41, and, as such, hasan amino acid sequence that includes SEQ ID NO:33, SEQ ID NO:37 and/orSEQ ID NO:40.

Preferred equine influenza PB1-N proteins of the present inventioninclude proteins encoded by a nucleic acid molecule comprisingnei_(wt1)PB1-N₁₂₂₉, nei_(wt1)PB1-N₁₁₉₄, nei_(wt2)PB1-N₆₇₃,nei_(wt2)PB1-N₆₃₆, nei_(ca1)PB21-N₁₂₂₅, nei_(ca1)PB1-N₁₁₈₅, and/ornei_(ca2)PB1-N₁₂₂₁. Preferred equine influenza PB1-N proteins arePei_(wt1)PB1-N₃₉₈, P_(wt2)PB1-N₂₁₂ and/or P_(ca1)PB1-N₃₉₅. In oneembodiment, a preferred equine influenza PB1-N protein of the presentinvention is encoded by SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:50, and/or SEQ ID NO:51, and, as such,has an amino acid sequence that includes SEQ ID NO:43, SEQ ID NO:46and/or SEQ ID NO:49.

Preferred equine influenza PB1-C proteins of the present inventioninclude proteins encoded by a nucleic acid molecule comprisingnei_(wt1)PB1-C₁₂₃₄, nei_(wt1)PB1-C₁₁₈₈, nei_(wt2)PB1-C₁₂₄₀,nei_(ca1)PB1-C₁₂₄₁, nei_(ca1)PB1-C₁₁₈₈, and/or nei_(ca2)PB1-C₁₂₄₁.Preferred equine influenza PB1-C proteins are Pei_(wt1)PB1-C₃₉₆,Pei_(wt2)PB1-C₃₉₆ Pei_(ca1)PB1-C₃₉₆, and/or Pei_(ca2)PB1-C₃₉₆. In oneembodiment, a preferred equine influenza PB1-C protein of the presentinvention is encoded by SEQ ID NO:53, SEQ ID NO:60, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, and/or SEQ ID NO:66, and, as such, has an aminoacid sequence that includes SEQ ID NO:54, SEQ ID NO:62, SEQ ID NO:64,and/or SEQ ID NO:67.

Preferred equine influenza PB1 proteins of the present invention includeproteins encoded by a nucleic acid molecule comprising nei_(wt)PB1₂₃₄₁,nei_(wt)PB1₂₂₇₁, nei_(ca1)PB1₂₃₄₁, nei_(ca1)PB1₂₂₇₁. Preferred equineinfluenza PB1 proteins are Pei_(wt)PB1₇₅₇, and/or Pei_(ca1)PB1₇₅₇. Inone embodiment, a preferred equine influenza PB1 protein of the presentinvention is encoded by SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:71, and/orSEQ ID NO:73, and, as such, has an amino acid sequence that includes SEQID NO:69 and/or SEQ ID NO:72.

Preferred equine influenza PA-C proteins of the present inventioninclude proteins encoded by a nucleic acid molecule comprisingnei_(wt1)PA-C₁₂₂₈, nei_(wt1)PA-C₁₁₆₄, nei_(wt2)PA-C₁₂₂₃,nei_(ca1)PA-C₁₂₃₃, and/or nei_(ca1)PA-C₁₁₇₀. Preferred equine influenzaPA-C proteins are Pei_(wt1)PA-C₃₈₈, and/or Pei_(ca1)PA-C₃₉₀. In oneembodiment, a preferred equine influenza PA-C protein of the presentinvention is encoded by SEQ ID NO:76, SEQ ID NO:82, SEQ ID NO:83, SEQ IDNO:84, and/or SEQ ID NO:86, and, as such, has an amino acid sequencethat includes SEQ ID NO:77 and/or SEQ ID NO:85.

Preferred equine influenza PA-N proteins of the present inventioninclude proteins encoded by a nucleic acid molecule comprisingnei_(wt)PA-N₁₂₁₆, nei_(wt)PA-N₁₁₉₃, nei_(ca)PA-N₁₂₁₇ andnei_(ca)PA-N₁₁₉₃. Preferred equine influenza PA-N proteins arePei_(wt)PA-N₃₉₇ and/or Pei_(ca)PA-N₃₉₇. In one embodiment, a preferredequine influenza PA-N protein of the present invention is encoded by SEQID NO:94, SEQ ID NO:95, SEQ ID NO:97, and/or SEQ ID NO:98, and, as such,has an amino acid sequence that includes SEQ ID NO:96 and/or SEQ IDNO:99.

Preferred equine influenza PA proteins of the present invention includeproteins encoded by a nucleic acid molecule comprising nei_(wt)PA₁₂₄₈and nei_(ca)PA₁₂₄₈. Preferred equine influenza PA proteins arePei_(wt)PA₇₁₆ and/or Pei_(ca)PA₇₁₆. In one embodiment, a preferredequine influenza PA protein of the present invention is encoded by SEQID NO:100 and/or SEQ ID NO:102, and, as such, has an amino acid sequencethat includes SEQ ID NO:101 and/or SEQ ID NO:103.

Preferred equine influenza NA proteins of the present invention includeproteins encoded by a nucleic acid molecule comprising nei_(ca)NA₁₄₇₈and nei_(ca)NA₁₄₁₀. A preferred equine influenza NA protein isPei_(ca)NA₄₇₀. In one embodiment, a preferred equine influenza NAprotein of the present invention is encoded by SEQ ID NO:87 and/or SEQID NO:89, and, as such, has an amino acid sequence that includes SEQ IDNO:88.

The present invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a PB2-N protein having anamino acid sequence comprising SEQ ID NO:9. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a PB2-C protein having anamino acid sequence comprising SEQ ID NO:22. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a PB2 protein having anamino acid sequence comprising SEQ ID NO:28. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a NS protein having anamino acid sequence comprising SEQ ID NO:40. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a PB1-N protein having anamino acid sequence comprising SEQ ID NO:49. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a PA-C protein having anamino acid sequence comprising SEQ ID NO:85. Another embodiment of thepresent invention includes a nucleic acid molecule comprising acold-adapted equine influenza virus encoding a PB1-C protein having anamino acid sequence comprising SEQ ID NO:64 and/or SEQ ID NO:67. Anotherembodiment of the present invention includes a nucleic acid moleculecomprising a cold-adapted equine influenza virus encoding a PB1 proteinhaving an amino acid sequence comprising SEQ ID NO:72. Anotherembodiment of the present invention includes a nucleic acid moleculecomprising a cold-adapted equine influenza virus encoding a PA-N proteinhaving an amino acid sequence comprising SEQ ID NO:99. Anotherembodiment of the present invention includes a nucleic acid moleculecomprising a cold-adapted equine influenza virus encoding a PA proteinhaving an amino acid sequence comprising SEQ ID NO:103. Anotherembodiment of the present invention includes a nucleic acid moleculecomprising a cold-adapted equine influenza virus encoding a NA proteinhaving an amino acid sequence comprising SEQ ID NO:88. It should benoted that since nucleic acid sequencing technology is not entirelyerror-free, the nucleic acid sequences and amino acid sequencespresented herein represent, respectively, apparent nucleic acidsequences of nucleic acid molecules of the present invention andapparent amino acid sequences of PB2-N, PB2-C, PB2, NS, PB1-N, PB1-C,PB1, PA-C, PA-N, PA, and NA proteins of the present invention.

Another embodiment of the present invention is an antibody thatselectively binds to an wild-type virus PB2-N, PB2-C, PB2, NS, PB1-N,PB1-C, PB1, PA-C, PA-N, PA and NA protein of the present invention.Another embodiment of the present invention is an antibody thatselectively binds to a cold-adapted virus PB2-N, PB2-C, PB2, NS, PB1-N,PB1-C, PB1, PA-C, PA-N, PA and NA protein of the present invention.Preferred antibodies selectively bind to SEQ ID NO:6, SEQ ID NO:9, SEQID NO:14, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:33, SEQ IDNO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ IDNO:54, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:72, SEQ ID NO:77, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:96, SEQ IDNO:99, SEQ ID NO:101 and SEQ ID NO:103.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLE 1

This example describes the cloning and sequencing of equine influenzaPB2 protein (RNA-directed RNA polymerase) nucleic acid moleculescorresponding to the N-terminal portion of the protein, for wild type orcold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PB2-N proteins were produced as follows. A PCR productcontaining a N-terminal portion of the equine PB2 gene was produced byPCR amplification from equine influenza virus DNA, and primers w570 andw571, designated SEQ ID NO:36 and SEQ ID NO:37, respectively. A nucleicacid molecule of 1241 nucleotides encoding a wild type PB2-N protein,denoted nei_(wt)PB2-N₁₂₄₁, with a coding strand having a nucleic acidsequence designated SEQ ID NO:13 was produced by further PCRamplification using the above described PCR product as a template andcloned into pCR 2.1®TA cloning vector, available from Invitrogen,Carlsbad, Calif., using standard procedures recommended by themanufacturer. The primers used were the T7 primer, designated by SEQ IDNO:29 and the REV primer, designated by SEQ ID NO:28. Plasmid DNA waspurified using a mini-prep method available from Qiagen, Valencia,Calif. PCR products were prepared for sequencing using a PRISM™ DyeTerminator Cycle Sequencing Ready Reaction kit, a PRISM™ dRhodamineTerminator Cycle Sequencing Ready Reaction kit, or a PRISM™ BigDye™Terminator Cycle Sequencing Ready Reaction kit, all available from PEApplied Biosystems, Foster City, Calif., following the manufacturer'sprotocol. Specific PCR conditions used with the kit were a rapid ramp to95° C., hold for 10 seconds followed by a rapid ramp to 50° C. with a 5second hold then a rapid ramp to 60° C. with a 4 minute hold, repeatingfor 25 cycles. T7 and REV primers were used in one reaction. PCRproducts were purified by ethanol/magnesium chloride precipitation.Automated sequencing of DNA samples was performed using an ABI PRISM™Model 377 with XL upgrade DNA Sequencer, available from PE AppliedBiosystems.

Translation of SEQ ID NO:13 indicates that nucleic acid moleculenei_(wt)PB2-N₁₂₄₁ encodes a N-terminal portion of influenza PB2 proteinof about 404 amino acids, referred to herein as P_(wt)PB2-N₄₀₄, havingamino acid sequence SEQ ID NO:14, assuming an open reading frame inwhich the initiation codon spans from nucleotide 28 through nucleotide30 of SEQ ID NO:13, and the last codon spans from nucleotide 1237through nucleotide 1239. The region encoding P_(wt)PB2-N₄₀₄, designatednei_(wt)PB2-N₁₂₁₄, and having a coding strand comprising nucleotides 28to 1239 of SEQ ID NO:13 is represented by SEQ ID NO:15.

B. A nucleic acid molecule of 1239 nucleotides encoding a N-terminalportion of influenza PB2 cold-adapted equine influenza virus PB2-Nprotein, denoted nei_(ca1)PB2-N₁₂₄₁, with a coding strand having asequence designated SEQ ID NO:16 was produced, and sequenced asdescribed in part A.

Translation of SEQ ID NO:16 indicates that nucleic acid moleculenei_(ca1)PB2-N₁₂₄₁ encodes a—terminal portion of equine influenza PB-2protein of about 404 amino acids, referred to herein as P_(ca1)PB2-N₄₀₄,having amino acid sequence SEQ ID NO:17, assuming an open reading framein which the initiation codon spans from nucleotide 28 throughnucleotide 30 of SEQ ID NO:16, and the last codon spans from nucleotide1237 through nucleotide 1239. The region encoding P_(ca1)PB2-N₄₀₄,designated nei_(ca1)PB₂-N₁₂₁₄, and having a coding strand comprisingnucleotides 28 to 1239 of SEQ ID NO:16, is represented by SEQ ID NO:18.

PCR amplification of a second nucleic acid molecule encoding acold-adapted equine influenza PB2-N protein in the same manner resultedin molecules nei_(ca2)PB2-N₁₂₄₁, identical to nei_(ca1)PB2-N₁₂₄₁, andnei_(ca2)PB2-N₁₂₁₄, identical to nei_(ca1)PB2-N₁₂₁₄.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt)PB2-N₁₂₄₁ (SEQ ID NO:13) and nei_(ca1)PB2-N₁₂₄₁ (SEQ ID NO:16)by DNA alignment reveals the following difference: a T to C base shiftat base 370. Comparison of the amino acid sequences of proteinsP_(wt)PB2-N₄₀₄ (SEQ ID NO:14) and P_(ca1)PB2-N₄₀₄ (SEQ ID NO:17) revealsthe following difference: a Y to H shift at amino acid 124 relating tothe a T to C shift at base 370 in the DNA sequence.

EXAMPLE 2

This example describes the cloning and sequencing of equine influenzaPB2 protein (RNA-directed RNA polymerase) nucleic acid moleculescorresponding to the C-terminal portion of the protein, for wild type orcold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PB2-C proteins were produced as follows. A PCR productcontaining the C-terminal portion of the equine PB2 gene was produced byPCR amplification using from equine influenza virus DNA and primers w572and w573, designated SEQ ID NO:38 and SEQ ID NO:39, respectively. Anucleic acid molecule of 1233 nucleotides encoding a wild type PB2-Cprotein, denoted nei_(wt)PB2-C₁₂₃₃, with a coding strand having anucleic acid sequence designated SEQ ID NO:19 was produced by furtherPCR amplification using the above-described PCR product as a templateand cloned as described in Example 1B. Plasmid DNA was purified andsequenced as in Example 1A, except that different primers were used inthe sequencing kits. T7 and REV were used in one instance; efPB2-a1,designated SEQ ID NO:40 and efPB2-s1, designated SEQ ID NO:41 were usedin another instance, and efPB2-a2, designated SEQ ID NO:42 and efPB2-s2,designated SEQ ID NO:43 were used in another instance.

Translation of SEQ ID NO:19 indicates that nucleic acid moleculenei_(wt1)PB2-C₁₂₃₃ encodes a C-terminal portion of influenza PB2 proteinof about 398 amino acids, referred to herein as P_(wt)PB2-C₃₉₈, havingamino acid sequence SEQ ID NO:20, assuming an open reading frame havinga first codon spans from nucleotide 3 through nucleotide 5 and atermination codon which spans from nucleotide 1197 through nucleotide1199 of SEQ ID NO:19. Because SEQ ID NO:19 is only a partial genesequence, it does not contain an initiation codon. The region encodingP_(wt)PB2-C₃₉₈, designated nei_(wt)PB2-C₁₁₉₄, and having a coding strandcomprising nucleotides 3 to 1196 of SEQ ID NO:19 is represented by SEQID NO:21.

PCR amplification of a second nucleic acid molecule encoding a wild typeequine influenza PB2-N protein in the same manner resulted in a nucleicacid molecule of 1232 nucleotides denoted nei_(wt2)PB2-N₁₂₃₂ with acoding strand with a sequence designated SEQ ID NO:22.nei_(wt2)PB2-N₁₂₃₂ is identical to nei_(wt1)PB2-C₁₂₃₃, expect thatnei_(wt2)PB2-N₁₂₃₂ lacks one nucleotide on the 5′-end. Translation ofSEQ ID NO:22 indicates that nucleic acid molecule nei_(wt1)PB2-C₁₂₃₃also encodes P_(wt)PB2-C₃₉₈ (SEQ ID NO:20), assuming an open readingframe having a first codon which spans from nucleotide 2 throughnucleotide 4 and a termination codon spans from nucleotide 1196 throughnucleotide 1198 of SEQ ID NO:22. Because SEQ ID NO:22 is only a partialgene sequence, it does not contain an initiation codon. The nucleic acidmolecule having a coding strand comprising nucleotides 2 to 1195 of SEQID NO:22, denoted nei_(wt2)PB2-C₁₁₉₄, is identical to SEQ ID NO:21.

B. A nucleic acid molecule of 1232 nucleotides encoding a C-terminalportion of influenza PB2 cold-adapted equine influenza virus protein,denoted nei_(ca1)PB2-C₁₂₃₂, and having a coding strand having a sequencedesignated SEQ ID NO:23 was produced as described in part A, except thatthe pCR®-Blunt cloning vector was used.

Translation of SEQ ID NO:23 indicates that nucleic acid moleculenei_(ca1)PB2-C₁₂₃₂ encodes a C-terminal portion of equine influenza PB-2protein of about 398 amino acids, referred to herein as P_(ca1)PB2-C₃₉₈,having amino acid sequence SEQ ID NO:24, assuming an open reading framehaving a first codon which spans from nucleotide 2 through nucleotide 4and a termination codon spans from nucleotide 1196 through nucleotide1198 of SEQ ID NO:23. Because SEQ ID NO:23 is only a partial genesequence, it does not contain an initiation codon. The region encodingP_(ca1)PB2-C₃₉₈, designated nei_(ca1)PB2-C1194, and having a codingstrand comprising nucleotides 2 to 1195 of SEQ ID NO:23, is representedby SEQ ID NO:25.

PCR amplification of a second nucleic acid molecule encoding acold-adapted equine influenza PB2-C protein in the same manner resultedin molecules nei_(ca2)PB2-C₁₂₃₁, containing one less nucleotide at the3′end than nei_(ca1)PB2-N₁₂₄₁; and nei_(ca2)PB2-N₁₂₁₄, identical tonei_(ca1)PB2-N₁₂₁₄.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt1)PB2-C₁₂₃₃ (SEQ ID NO:19) and nei_(ca1)PB2-C₁₂₃₂ (SEQ ID NO:23)by DNA alignment reveals the following differences: an A to C base shiftat base 153 of SEQ ID NO:19, and a G to A base shift at base 929 of SEQID NO:19. Comparison of the amino acid sequences of proteinsP_(wt)PB2-C₃₉₈ (SEQ ID NO:20) and P_(ca1)PB2-₃₉₈ (SEQ ID NO:24) revealsthe following difference: a K to Q shift at amino acid 51 when relatingto the an A to C base shift at base 153 in the DNA sequences. There isno amino acid shift resulting from the G to A base shift at base 929.

EXAMPLE 3

This example describes the cloning and sequencing of equine influenzaPB2 protein (RNA-directed RNA polymerase) nucleic acid molecules forwild type or cold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PB2 proteins were produced as follows. The wild type orcold-adapted equine influenza genes were cloned in two fragments, theN-terminal portion was produced as in Example 1 and the C-terminalportion of the gene was produced as in Example 2. The DNA sequence forthe wild type equine influenza PB2 gene was generated by combining theconsensus sequences for the wild type PB2-N protein, denotednei_(wt)PB2-N₁₂₄₁, (SEQ ID NO:13) with the gene fragments for the wildtype PB2-C protein, denoted nei_(wt1)PB2-C₁₂₃₃ (SEQ ID NO:19) andnei_(wt2)PB2-C₁₂₃₂ (SEQ ID NO:22). The result of combining the consensussequences from the N-terminal and C-terminal portions of the PB2 wildtype influenza virus yielded a complete DNA sequence denotednei_(wt)PB2₂₃₄₁ (SEQ ID NO:44). Translation of SEQ ID NO:44 indicatesthat the nucleic acid molecule nei_(wt)PB2₂₃₄₁ encodes a full lengthequine influenza PB2 protein of about 759 amino acids referred to hereinas Pei_(wt)PB2₇₅₉, having amino acid sequence SEQ ID NO: 45, assuming anopen reading frame in which the initiation codon spans from nucleotide28 through nucleotide 30 of SEQ ID NO: 44 and the termination codonspans from nucleotide 2305 through nucleotide 2307 of SEQ ID NO: 44. Theregion encoding Pei_(wt)PB2₇₅₉, designated nei_(wt)PB2₂₂₇₇, and having acoding strand comprising nucleotides 28 to 2304 of SEQ ID NO: 44, is SEQID NO: 46.

B. A DNA sequence of 2341 nucleotides encoding a cold-adapted equineinfluenza virus PB2, denoted nei_(ca1)PB2₂₃₄₁, with a sequence denotedSEQ ID NO: 47 was produced by combining the sequences for the N-terminaland C-terminal portions of the PB2 cold-adapted equine influenza gene.The clones for the N-terminal sequences are denoted nei_(ca1)PB2-N₁₂₄₁,and nei_(ca2)PB2-N₁₂₄₁, which are identical and are represented by SEQID NO:16. The clones for the C-terminal sequences are denotednei_(ca1)PB2-C₁₂₃₂ and nei_(ca2)PB2-C₁₂₃₁, represented by SEQ ID NO:23.

Translation of SEQ ID NO:47 indicates that nucleic acid moleculenei_(ca1)PB2₂₃₄₁ encodes a full-length equine influenza PB2 protein ofabout 759 amino acids, referred to herein as Pei_(ca1)PB2₇₅₉, havingamino acid sequence SEQ ID NO:48, assuming an open reading frame inwhich the initiation codon spans from nucleotide 28 through nucleotide30 of SEQ ID NO: 47 and the termination codon spans from nucleotide 2305through nucleotide 2307 of SEQ ID NO:47. The region encodingPei_(ca1)PB2₇₅₉ designated nei_(ca1)PB2₂₂₇₇ and having a coding strandcomprising nucleotides 28 to 2304 of SEQ ID NO:49.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt)PB₂₃₄₁ (SEQ ID NO:44) and nei_(ca1)PB2₂₃₄₁ (SEQ ID NO:47) by DNAalignment reveals the following differences: a T to C base shift at base370, a A to C base shift at base 1261, and a G to A base shift at base2037. Comparison of the amino acid sequences of proteins Pei_(wt)PB2₇₅₉(SEQ ID NO:45) and Pei_(ca1)PB2₇₅₉ (SEQ ID NO:48) reveals the followingdifferences: a Y to H shift at amino acid 124 relating to the a T to Cshift at base 370 in the DNA sequence, a K to Q shift at amino acid 421relating to the A to C shift at base 1261 in the DNA sequence. The thirdnucleotide shift at base 2037 does not result in an amino acid shift.

EXAMPLE 4

This example describes the cloning and sequencing of equine influenza NS(nonstructural) protein nucleic acid molecules for wild type orcold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus NS proteins were produced as follows. A PCR productcontaining an equine NS gene was produced by PCR amplification fromequine influenza virus DNA and primers w586 and w587, designated SEQ IDNO:59 and SEQ ID NO:60, respectively. A nucleic acid molecule of 891nucleotides encoding a wild-type NS protein, denoted nei_(wt)NS₈₉₁, witha coding strand having a nucleic acid sequence designated SEQ ID NO:50was produced by further PCR amplification using the above-described PCRproduct as a template and cloned into pCR 2.1®TA cloning vector asdescribed in Example 1A. Plasmid DNA was purified and sequenced as inExample 1A, except that primers used in the sequencing kits were only T7and REV.

Translation of SEQ ID NO:50 indicates that nucleic acid moleculenei_(wt1)NS₈₉₁ encodes a full-length equine influenza NS protein ofabout 230 amino acids, referred to herein as Pei_(wt1)NS₂₃₀, havingamino acid sequence SEQ ID NO:51, assuming an open reading frame inwhich the initiation codon spans from nucleotide 27 through nucleotide29 of SEQ ID NO:50 and the termination codon spans from nucleotide 717through nucleotide 719 of SEQ ID NO:50. The region encodingPei_(wt1)NS₂₃₀, designated nei_(wt1)NS₆₉₀, and having a coding strandcomprising nucleotides 27 to 716 of SEQ ID NO:50 is represented by SEQID NO:52.

PCR amplification of a second nucleic acid molecule encoding a wild typeequine influenza NS protein in the same manner resulted in moleculesnei_(wt2)NS₈₉₁, identical to nei_(wt1)NS₈₉₁, in the coding region, i.e.nei_(wt2)NS₆₉₀, is identical to nei_(wt1)NS₆₉₀. nei_(wt2)NS₈₉₁ differsfrom nei_(wt1)NS₈₉₁ in one nucleotide at base 827 (G to A) which is 111bases downstream from the stop codon. PCR amplification of a thirdnucleic acid encoding a wild type equine influenza NS protein in thesame manner resulted in a nucleic acid molecule of 888 nucleotidesdenoted nei_(wt3)NS₈₈₈, with a coding strand with a nucleic acidsequence designated SEQ ID NO: 53. nei_(wt3)NS₈₈₈ is identical tonei_(wt1)NS₈₉₁, except that nei_(wt3)NS₈₈₈, lacks two nucleotides on the5′ end and one nucleotide on the 3′ end. Translation of SEQ ID NO:53indicates that nucleic acid molecule nei_(wt3)NS₈₈₈ also encodesPei_(wt1)NS₂₃₀ (SEQ ID NO:51), assuming an open reading frame having aninitiation codon which spans from nucleotide 25 through nucleotide 27 ofSEQ ID NO:53 and a termination codon which spans from nucleotide 715through nucleotide 717 of SEQ ID NO:53. The nucleic acid molecule havinga coding strand comprising nucleotides 25 to 714 of SEQ ID 53, denotednei_(wt3)NS₆₉₀, is identical to SEQ ID NO:52.

PCR amplification of a fourth nucleic acid of 468 nucleotides encoding aC-terminal portion of the wild type equine influenza NS protein, denotednei_(wt4)NS468 and having a coding sequence designated SEQ ID NO:54 wasproduced. Translation of SEQ ID NO:54 indicates that nucleic acidmolecule nei_(wt4)NS₄₆₈ encodes a C-terminal portion of equine influenzaNS protein of about 97 amino acids, referred to herein as Pei_(wt4)NS₉₇,having amino acid sequence SEQ ID NO:55, assuming an open reading framehaving a first codon which spans from nucleotide 3 to 5 of SEQ ID NO:54, and a termination codon spans from nucleotide 294 through 296 of SEQID NO:54. Because SEQ ID NO:54 is only a partial gene sequence, it doesnot contain an initiation codon. The region encoding Pei_(wt4)NS₉₇,designated nei_(wt4)NS₂₉₃, and having a coding strand comprisingnucleotides 1 to 293 of SEQ ID NO:54, is represented by SEQ ID NO: 56.

B. A nucleic acid molecule of 888 nucleotides encoding a cold-adaptedequine influenza virus NS protein, denoted nei_(ca1)NS₈₈₈, with a codingstrand having a sequence designated SEQ ID NO:57 was produced andsequenced as described in part A.

Translation of SEQ ID NO:57 indicates that nucleic acid moleculenei_(ca1)NS₈₈₈ encodes a full-length equine influenza NS protein ofabout 230 amino acids, referred to herein as Pei_(ca1)NS₂₃₀, havingamino acid sequence SEQ ID NO:58, assuming an open reading frame inwhich the initiation codon spans from nucleotide 27 through nucleotide29 of SEQ ID NO:57 and the termination codon spans from nucleotide 717through nucleotide 719 of SEQ ID NO:57. The region encodingPei_(ca1)NS₂₃₀, designated nei_(ca1)NS₆₉₀, and having a coding strandcomprising nucleotides 27 to 716 of SEQ ID NO:57, is represented by SEQID NO:59.

PCR amplification of a second nucleic acid molecule encoding acold-adapted equine influenza NS protein in the same manner resulted inmolecules nei_(ca2)NS₈₈₇, containing one less nucleotide at the 3′ endthan nei_(ca1)NS₈₈₈; the coding region nei_(ca2)NS690 is identical tonei_(ca1)NS₆₉₀.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt)NS₈₉₁ (SEQ ID NO:50) and nei_(ca1)NS₈₈₈ (SEQ ID NO:57) by DNAalignment reveals the following difference: a A to G shift at base 827which is 111 bases downstream from the stop codon. The 3′ fragmentencoding nei_(wt4)NS₄₆₈ (SEQ ID NO:54) has one shift T to C found atbase 633 relative to the full-length consensus sequence. Comparison ofthe amino acid sequences of proteins Pei_(wt)NS₂₃₀ (SEQ ID NO:51) andPei_(ca1)NS₂₃₀ (SEQ ID NO:58) reveals that there are no differencesbetween amino acid sequences of the wild type and cold-adapted proteins.

EXAMPLE 5

This example describes the cloning and sequencing of equine influenzaPB1 protein (RNA-directed RNA polymerase 1) nucleic acid moleculescorresponding to the N-terminal portion of the protein, for wild type orcold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PB1-N proteins were produced as follows. A PCR productcontaining a N-terminal portion of the equine PB1 gene was produced byPCR amplification from equine influenza virus DNA, and primers T7 andREV. A nucleic acid molecule of 1229 nucleotides encoding a wild typePB1-N protein, denoted nei_(wt1)PB1-N₁₂₂₉, with a coding strand having anucleic acid sequence designated SEQ ID NO:62 was produced by furtherPCR amplification using the above described PCR product as a templateand cloned as described in Example 1B. Plasmid DNA was purified andsequenced as in Example 1B, except that only T7 and REV primers wereused in the sequencing kits.

Translation of SEQ ID NO:62 indicates that nucleic acid moleculenei_(wt1)PB1-N₁₂₂₉ encodes a N-terminal portion of influenza PB1 proteinof about 398 amino acids, referred to herein as Pei_(wt1)PB1-N₃₉₈,having amino acid sequence SEQ ID NO:63, assuming an open reading framein which the initiation codon spans from nucleotide 36 throughnucleotide 38 of SEQ ID NO:62, and the last codon spans from nucleotide1227 through nucleotide 1229 of SEQ ID NO:62. The region encodingPei_(wt1)PB1-N₃₉₈, designated nei_(wt1)PB1-N₁₁₉₄, and having a codingstrand comprising nucleotides 36 to 1229 of SEQ ID NO:62 is representedby SEQ ID NO:64.

PCR amplification of a second nucleic acid molecule encoding a wild typeequine influenza PB1-N protein in the same manner resulted in a nucleicacid molecule of 673 nucleotides denoted nei_(wt2)PB1-N₆₇₃, with acoding strand with a sequence designated SEQ ID NO:65. Translation ofSEQ ID NO:65 indicates that nucleic acid molecule nei_(wt2)PB1-N₆₇₃encodes Pei_(wt2)PB1-N₂₁₂ (SEQ ID NO:66), assuming an open reading framehaving an initiation codon which spans from nucleotide 36 throughnucleotide 38 of SEQ ID NO:65 and a last codon which spans fromnucleotide 671 through nucleotide 673 of SEQ ID NO:65. Because SEQ IDNO:65 is only a partial gene sequence, it does not contain a stop codon.The nucleic acid molecule having a coding strand comprising nucleotides36 to 671 of SEQ ID NO:65, denoted nei_(wt2)PB1-N₆₃₆, is designated SEQID NO:67.

B. A nucleic acid molecule of 1225 nucleotides encoding a N-terminalportion of influenza PB1 cold-adapted equine influenza virus PB1-Nprotein, denoted nei_(ca1)PB1-N₁₂₂₅, with a coding strand having asequence designated SEQ ID NO:68 was produced, and sequenced asdescribed in part A.

Translation of SEQ ID NO:68 indicates that nucleic acid moleculenei_(ca1)PB1-N₁₂₂₅ encodes a N-terminal portion of equine influenza PB-1protein of about 395 amino acids, referred to herein asPei_(ca1)PB1-N₃₉₅, having amino acid sequence SEQ ID NO:69, assuming anopen reading frame in which the initiation codon spans from nucleotide34 through nucleotide 36 of SEQ ID NO:68, and a last codon which spansfrom nucleotide 1216 through nucleotide 1218 of SEQ ID NO:68. The regionencoding Pei_(ca1)PB1-N₃₉₅, designated nei_(ca1)PB1-N₁₁₈₅, and having acoding strand comprising nucleotides 34 to 1218 of SEQ ID NO:68, isrepresented by SEQ ID NO:70.

PCR amplification of a second nucleic acid molecule encoding acold-adapted equine influenza PB1-N protein in the same manner resultedin molecules nei_(ca2)PB1-N₁₂₂₁, designated SEQ ID NO:71, containingfour less nucleotides at the 5′ end than nei_(ca1)PB1-N₁₂₂₅; the codingregion nei_(ca2)PB1-N₁₁₈₅, is identical to nei_(ca1)PB1-N₁₁₈₅.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt)PB1-N₁₂₂₉ (SEQ ID NO:62) and nei_(ca1)PB1-N₁₂₂₅ (SEQ ID NO:68)by DNA alignment reveals no differences in the coding regions.Comparison of the amino acid sequences of proteins Pei_(wt)PB1-N₃₉₅ (SEQID NO:63) and Pei_(ca1)PB1-N₃₉₅ (SEQ ID NO:69) also reveals nodifferences.

EXAMPLE 6

This example describes the cloning and sequencing of equine influenzaPB1 protein (RNA-directed RNA polymerase 1) nucleic acid moleculescorresponding to the C-terminal portion of the protein, for wild type orcold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PB1-C proteins were produced as follows. A PCR productcontaining an C-terminal portion of the equine PB1 gene was produced byPCR amplification from equine influenza virus DNA, and primer w569designated SEQ ID NO:102. A nucleic acid molecule of 1234 nucleotidesencoding a wild type PB1-C protein, denoted nei_(wt1)PB1-C₁₂₃₄, with acoding strand having a nucleic acid sequence designated SEQ ID NO:85 wasproduced by further PCR amplification using the above described PCRproduct as a template and cloned as described in Example 1B. Plasmid DNAwas purified and sequenced as in Example 1A, except that differentprimers were used in the sequencing kits. T7, REV, w569, efPB1-a1,designated SEQ ID NO:97, efPB1-a2, designated SEQ. ID NO:98, efPB1-s1,designated SEQ ID NO: 99, efPB1-s2, designated SEQ ID NO: 100, andefPB1-s3, designated SEQ ID NO:101 were used in one instance, T7, REV,efPB1-a1, efPB1-a2, efPB1-s1, efPB1-s2, and efPB1-s3 were used inanother instance and T7 and REV were used in another instance.

Translation of SEQ ID NO:85 indicates that nucleic acid moleculenei_(wt1)PB1-C₁₂₃₄ encodes an C-terminal portion of influenza PB1protein of about 396 amino acids, referred to herein asPei_(wt1)PB1-C₃₉₆, having amino acid sequence SEQ ID NO:86, assuming anopen reading frame in which the first codon spans from nucleotide 1through nucleotide 3 of SEQ ID NO:85 and a termination codon which spansfrom nucleotide 1189 through nucleotide 1191 of SEQ ID NO:85. BecauseSEQ ID NO:85 is only a partial gene sequence, it does not contain aninitiation codon. The region encoding Pei_(wt1)PB1-C₃₉₆, designatednei_(wt1)PB1-C₁₁₈₈, and having a coding strand comprising nucleotides 1to 1188 of SEQ ID NO:85 is represented by SEQ ID NO:87.

PCR amplification of a second nucleic acid molecule encoding a wild typeequine influenza PB1-C protein in the same manner resulted in a nucleicacid molecule of 1240 nucleotides denoted nei_(wt2)PB1-C₁₂₄₀, with acoding strand with a sequence designated SEQ ID NO:88. Translation ofSEQ ID NO:88 indicates that nucleic acid molecule nei_(wt2)PB1-N₁₂₄₀encodes a molecule designated Pei_(wt2)PB1-C₃₉₆ (SEQ ID NO: 89) whichdiffers from Pei_(wt1)PB1-C₃₉₆ (SEQ ID NO:85) in one nucleotide.Nucleotide 382 of nei_(wt1)PB1-C₁₂₃₄, i.e. nucleotide 382 ofnei_(wt1)PB1-C₁₁₈₈ was A, while nucleotide 389 of nei_(wt2)PB1-C₁₂₄₀,i.e. nucleotide 382 of nei_(wt2)PB1-C₁₁₈₈ was T. Translation ofnei_(wt2)PB1-C₁₂₄₀ results in an amino acid change of T to S.

B. A nucleic acid molecule of 1241 nucleotides encoding an C-terminalportion of influenza PB1 cold-adapted equine influenza virus PB1-Cprotein, denoted nei_(ca1)PB1-C₁₂₄₁, with a coding strand having asequence designated SEQ ID NO:91 was produced, and sequenced asdescribed in part A.

Translation of SEQ ID NO:91 indicates that nucleic acid moleculenei_(ca1)PB1-C₁₂₄₁ encodes an C-terminal portion of equine influenzaPB-1 protein of about 396 amino acids, referred to herein asPei_(ca1)PB1-C₃₉₆, having amino acid sequence SEQ ID NO:92, assuming anopen reading frame in which the first codon spans from nucleotide 8through nucleotide 10 of SEQ ID NO:91 and a termination codon that spansfrom nucleotide 1196 through nucleotide 1198 of SEQ ID NO:91. BecauseSEQ ID NO:91 is only a partial gene sequence, it does not contain aninitiation codon. The region encoding Pei_(ca1)PB1-C₃₉₆, designatednei_(ca1)PB1-C₁₁₈₈, and having a coding strand comprising nucleotides 8to 1195 of SEQ ID NO:91, is represented by SEQ ID NO:93.

PCR amplification of a second nucleic acid molecule encoding acold-adapted equine influenza PB1-C protein in the same manner resultedin a nucleic acid molecule of 1241 nucleotides denotednei_(ca2)PB1-C₁₂₄₁, with a coding strand with a sequence designated SEQID NO:94. Translation of SEQ ID NO:94 indicates that nucleic acidmolecule nei_(ca2)PB1-C₁₂₄₁ encodes a molecule designatedPei_(ca2)PB1-C₃₉₆ (SEQ ID NO:95) which differs from Pei_(ca1)PB1-C₃₉₆(SEQ ID NO:92) in one nucleotide. Nucleotide 1044 of nei_(ca1)PB1-C₁₂₄₁,i.e. nucleotide 1037 of nei_(ca1)PB1-N₁₁₈₈ was A, while nucleotide 1044of nei_(ca2)PB1-C₁₂₄₁, i.e. nucleotide 1037 of nei_(ca2)PB1-C₁₁₈₈ was G.Translation of nei_(ca2)PB1-C₁₂₄₁ results in an amino acid change of Rto K.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt1)PB1-C₁₂₃₄ (SEQ ID NO:85) and nei_(ca1)PB1-C₁₂₄₁ (SEQ ID NO:91)by DNA alignment reveals the following differences: a C to T shift atbase 600 of SEQ ID NO:85, and a T to A shift at base 603 of SEQ IDNO:85. Comparison of the amino acid sequences of proteinsPei_(wt1)PB1-C₃₉₆ (SEQ ID NO:86) and Pei_(ca1)PB1-N₃₉₆ (SEQ ID NO:92)reveals the following difference: a H to Q amino acid shift 203 whenrelating to the T to A base shift at base 603 in the DNA sequences.There is no amino acid shift resulting from the C to T base shift atbase 600.

EXAMPLE 7

This example describes the cloning and sequencing of equine influenzaPB1 protein (RNA-directed RNA polymerase) nucleic acid molecules forwild type or cold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PB1 proteins were produced as follows. The wild type orcold-adapted equine influenza genes were cloned in two fragments, theN-terminal portion was produced as in Example 5 and the C-terminalportion of the gene was produced as in Example 6.

The DNA sequence for the wild type equine influenza PB1 gene wasgenerated by combining the sequences for the wild type PB1-N protein,nei_(wt1)PB1-N₁₂₂₉ (SEQ ID NO:62) and nei_(wt2)PB1-N₆₇₃ (SEQ ID NO:65)with the gene fragments for the wild type PB1-C protein, denotednei_(wt1)PB1-C₁₂₃₄ (SEQ ID NO:85) and nei_(wt2)PB1-C₁₂₄₀ (SEQ ID NO:88). The result of combining the N-terminal and C-terminal portions ofthe PB1 wild type influenza virus yielded a complete DNA sequence of2341 nucleotides denoted nei_(wt)PB1₂₃₄₁ (SEQ ID NO:103). Translation ofSEQ ID NO:103 indicates that the nucleic acid molecule nei_(wt)PB2₂₃₄₁encodes a full length equine influenza PB1 protein of about 757 aminoacids referred to herein as Pei_(wt)PB1₇₅₇, having amino acid sequenceSEQ ID NO:104, assuming an open reading frame in which the initiationcodon spans from nucleotide 25 through nucleotide 27 of SEQ ID NO: 103and the termination codon spans from nucleotide 2293 through nucleotide2295 of SEQ ID NO: 103. The region encoding, Pei_(wt)PB1₇₅₇ designatednei_(wt)PB1₂₂₇₁, and having a coding strand comprising nucleotides 25 to2292 of SEQ ID NO: 103, is SEQ ID NO:105.

B. A DNA sequence of 2341 nucleotides encoding a cold-adapted equineinfluenza virus PB1, denoted nei_(ca1)PB1₂₃₄₁, with a sequence denotedSEQ ID NO: 106 was produced by combining the sequences for theN-terminal and C-terminal portions of the PB1 cold-adapted equineinfluenza gene. The clones for the N-terminal sequences are denotednei_(ca1)PB1-N₁₂₂₅ (SEQ ID NO: 68) and nei_(ca2)PB1-N₁₂₂₁ (SEQ ID NO:71). The clones for the C-terminal sequences are denotednei_(ca1)PB1-C₁₂₄₁ (SEQ ID NO:91) and nei_(ca2)PB1-C₁₂₄₁, (SEQ ID NO:94).

Translation of SEQ ID NO:106 indicates that nucleic acid moleculenei_(ca1)PB1₂₃₄₁ encodes a fill-length equine influenza PB1 protein ofabout 757 amino acids, referred to herein as Pei_(ca1)PB1₇₅₇, havingamino acid sequence SEQ ID NO:107, assuming an open reading frame inwhich the initiation codon spans from nucleotide 25 through nucleotide27 SEQ ID NO: 106 and the termination codon spans from nucleotide 2296through nucleotide 2298 of SEQ ID NO:106. The region encodingPei_(ca1)PB1₇₅₇ designated nei_(ca1)PB1₂₂₇₁ and having a coding strandcomprising nucleotides 25 to 2295 of SEQ ID NO:108.

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt)PB1₂₃₄₁ (SEQ ID NO:103) and nei_(ca1)PB1₂₃₄₁ (SEQ ID NO:106) byDNA alignment reveals the following differences: a C to T base shift atbase 1683, and a T to A base shift at base 1686. Comparison of the aminoacid sequences of proteins Pei_(wt)PB1₇₅₇ (SEQ ID NO:104) andPei_(ca1)PB1₇₅₇ (SEQ ID NO:107) reveals the following differences: noshift in base C at amino acid 561 relating to the C to T shift at base1683, and a H to Q shift at amino acid 562 relating to the a T to Ashift at base 1683 in the DNA sequence.

EXAMPLE 8

This example describes the cloning and sequencing of equine influenza PAprotein ( RNA polymerase A) nucleic acid molecules corresponding to theC-terminal portion of the protein, for wild type or cold-adapted equineinfluenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PA-C proteins were produced as follows. A PCR productcontaining the C-terminal portion of the equine PA gene was produced byPCR amplification using from equine influenza virus DNA and primers C+PAand C−PA, designated SEQ ID NO:83 and SEQ ID NO:84 respectively. Anucleic acid molecule of 1228 nucleotides encoding a wild type PA-Cprotein, denoted nei_(wt1)PA-C₁₂₂₈, with a coding strand having anucleic acid sequence designated SEQ ID NO:76 was produced by furtherPCR amplification using the above-described PCR product as a templateand cloned as described in Example 1B. Plasmid DNA was purified andsequenced as in Example 1A, except that different primers were used inthe sequencing kits. T7 and REV were used in one instance;PAC-1,designated SEQ ID NO:72, PAC-2, designated SEQ ID NO:73, PAC-3,designated SEQ ID NO:74, PAC-4, designated SEQ ID NO: 75, T7 and REVwere used in another instance; and PAC-1, PAC-2, T7 and REV were used inanother instance.

Translation of SEQ ID NO:76 indicates that nucleic acid moleculenei_(wt1)PA-C₁₂₂₈ encodes a C-terminal portion of influenza PA proteinof about 388 amino acids, referred to herein as Pei_(wt1)PA-C₃₈₈, havingamino acid sequence SEQ ID NO:77, assuming an open reading frame havinga first codon spans from nucleotide 3 through nucleotide 5 of SEQ IDNO:76 and a termination codon which spans from nucleotide 1167 throughnucleotide 1169 of SEQ ID NO:76. Because SEQ ID NO:76 is only a partialgene sequence, it does not contain an initiation codon. The regionencoding Pei_(wt1)PA-C₃₈₈, designated nei_(wt1)PA-C₁₁₆₄, and having acoding strand comprising nucleotides 3 to 1166 of SEQ ID NO:76 isrepresented by SEQ ID NO:78.

PCR amplification of a second nucleic acid molecule encoding a wild typeequine influenza PA-C protein in the same manner resulted in a nucleicacid molecule of 1223 nucleotides denoted nei_(wt2)PA-C₁₂₂₃, with acoding strand with a sequence designated SEQ ID NO:79. nei_(wt2)PA-C₁₂₂₃is identical to nei_(wt1)PA-C₁₂₂₈, with the exception of a T to C baseshift at base 753 and that nei_(wt2)PA-C₁₂₂₃ lacks five nucleotides onthe 3′-end. Translation of SEQ ID NO:79 indicates that nucleic acidmolecule nei_(wt2)PA-C₁₂₂₃ also encodes Pei_(wt1)PA-C₃₈₈ (SEQ ID NO:77),assuming an open reading frame having a first codon which spans fromnucleotide 3 through nucleotide 5 of SEQ ID NO:79 and a terminationcodon which spans from nucleotide 1167 through nucleotide 1169 of SEQ IDNO:79. Because SEQ ID NO:79 is only a partial gene sequence, it does notcontain an initiation codon. The nucleic acid molecule having a codingstrand comprising nucleotides 3 to 1166 of SEQ ID NO:79, denotednei_(wt2)PA-C₁₂₂₃, is identical to SEQ ID NO 78.

B. A nucleic acid molecule of 1233 nucleotides encoding a C-terminalportion of influenza PA-C cold-adapted equine influenza virus protein,denoted nei_(ca1)PA-C₁₂₃₃, and having a coding strand having a sequencedesignated SEQ ID NO:80 was produced as described in part A, except thatthe pCR®-Blunt cloning vector was used.

Translation of SEQ ID NO:80 indicates that nucleic acid moleculenei_(ca1)PA-C₁₂₃₃ encodes a C-terminal portion of equine influenza PAprotein of about 390 amino acids, referred to herein asPei_(ca1)PA-C₃₉₀, having amino acid sequence SEQ ID NO:81, assuming anopen reading frame having a first codon which spans from nucleotide 3through nucleotide 5 of SEQ ID NO:80 and a termination codon which spansfrom nucleotide 1173 through nucleotide 1175 of SEQ ID NO:80. BecauseSEQ ID NO:80 is only a partial gene sequence, it does not contain aninitiation codon. The region encoding Pei_(ca1)PA-C₃₉₀, designatednei_(ca1)PA-C₁₁₇₀, and having a coding strand comprising nucleotides 3to 1172 of SEQ ID NO:80, is represented by SEQ ID NO:82.

PCR amplification of a second nucleic acid molecule encoding acold-adapted equine influenza PA-C protein in the same manner resultedin molecule nei_(ca2)PA-C₁₂₃₃, containing one A to G base shift at base953 as compared to nei_(ca1)PA-C₁₂₃₃; this base shift does not result inan amino acid change so Pei_(ca2)PA-C₃₉₀, is identical toPei_(ca1)PA-C₃₉₀ (SEQ ID NO:81.)

C. Comparison of the nucleic acid sequences of the coding strands ofnei_(wt1)PA-C₁₂₂₈ (SEQ ID NO:76) and nei_(ca1)PA-C₁₂₃₃ (SEQ ID NO:80) byDNA alignment reveals the following difference: an C to T base shift atbase 753 of SEQ ID NO:80. Comparison of the amino acid sequences ofproteins Pei_(wt1)PA-C₃₈₈ (SEQ ID NO:77) and Pei_(ca1)PA-₃₉₀ (SEQ IDNO:81) reveals the following difference: a W to R shift at amino acid251 when relating to the C to T base shift at base 753 in the DNAsequences.

EXAMPLE 9

This example describes the cloning and sequencing of equine influenza PAprotein nucleic acid molecules corresponding to the N-terminal portionof the protein, for wild type or cold-adapted equine influenza viruses.

A. Nucleic acid molecules encoding wild type or cold-adapted equineinfluenza virus PA-N proteins were produced as follows. A PCR productcontaining the N-terminal portion of the equine influenza PA gene wasproduced using equine influenza virus DNA and primers REV (SEQ ID NO:1),T7 (SEQ ID NO:2), PAN-2 (SEQ ID NO:90), PAN-3 (SEQ ID NO:91), PAN-4 (SEQID NO:92), and PAN-5 (SEQ ID NO:93). Plasmid DNA was purified andsequenced as in Example 1.

A nucleic acid molecule of 1216 nucleotides encoding a wild type PA-Nprotein, denoted nei_(wt)PA-N₁₂₁₆, with a coding strand having a nucleicacid sequence designated SEQ ID NO:X5.

Translation of SEQ ID NO:X5 indicates that nucleic acid moleculenei_(wt)PA-N₁₂₁₆ encodes a N-terminal portion of influenza PA protein ofabout 397 amino acids, referred to herein as Pei_(wt)PA-N₃₉₇, havingamino acid sequence SEQ ID NO:X7, assuming an open reading frame fromthe starting methionine (M) through glutamic acid (E) at amino acid 397.The region encoding Pei_(wt)PA-N₃₉₇ designated nei_(wt)PA-N₁₁₉₃ andhaving a coding strand comprising nucleotides 24 to 1214 of SEQ IDNO:X5, is herein designated SEQ ID NO:X6.

B. A nucleic acid molecule of 1217 nucleotides encoding an N-terminalportion of influenza PA-N cold-adapted equine influenza virus protein,denoted nei_(ca)PA-N₁₂₁₇, with a coding strand having a nucleic acidsequence designated SEQ ID NO:X8.

Translation of SEQ ID NO:X9 indicates that nucleic acid moleculenei_(ca)PA-N₁₂₁₇ encodes a N-terminal portion of influenza PA protein ofabout 397 amino acids, referred to herein as Pei_(ca)PA-N₃₉₇, havingamino acid sequence SEQ ID NO:X10, assuming an open reading frame fromthe starting methionine (M) through glutamic acid (E) at amino acid 397.The region encoding Pei_(ca)PA-N₃₉₇, designated nei_(ca)PA-N₁₁₉₃ andhaving a coding strand comprising nucleotides 25 to 1215 of SEQ IDNO:X8, is designated herein as SEQ ID NO:X9.

C. Comparison of the amino acid sequences of proteins Pei_(wt)PA-N₃₉₇(SEQ ID NO:X7) and Pei_(ca)PA-N₃₉₇ (SEQ ID NO:X10) reveals the followingdifferences: a glutamic acid (E) to lysine (K) at amino acid 59 and analanine (A) to threonine (T) at amino acid 156.

EXAMPLE 10

This example describes the cloning and sequencing of equine influenza PAprotein nucleic acid molecules, for wild type or cold-adapted equineinfluenza viruses.

A. The nucleic acid sequences encoding wild type or cold-adapted equineinfluenza virus PA proteins were compiled as follows. The open readingframe DNA sequence is 2148 nucleotides long for both stains. There is a214 nucleotide overlap from the C-terminal and N-terminal fragmentsgenerated in Examples 8 and 9, respectively. The open reading framesequences encode the RNA Polymerase A, otherwise known as RNA Polymerase2 gene, which has 716 amino acid residues from the starting methionine(M) through lysine (K) at amino acid 716.

A nucleic acid molecule of 2148 nucleotides encoding the wild typeprotein is designed nei_(wt)PA₂₁₄₈, and has the coding strand of nucleicacid sequence SEQ ID NO:X11.

Translation of SEQ ID NO:X11 indicates that nucleic acid moleculenei_(wt)PA₂₁₄₈ encodes the equine influenza virus PA protein of about716 amino acids, referred to herein as Pei_(wt)PA₇₁₆, having amino acidsequence SEQ ID NO:X12.

B. A nucleic acid molecule of 2148 nucleotides encoding the cold-adaptedprotein is designated nei_(ca)PA₂₁₄₈, and has the coding strand ofnucleic acid sequence SEQ ID NO:X13.

Translation of SEQ ID NO:X13 indicates that nucleic acid moleculenei_(ca)PA₂₁₄₈ encodes a protein of about 716 amino acids, referred toherein as PEI_(ca)PA₇₁₆, having amino acid sequence SEQ ID NO:X14.

C. Comparison of the wild type and cold-adapted virus sequences revealsthree discrepancies: a G to A base shift at base 175, base 466, and base870. The base change at base 175 results in a glutamic acid (E) tolysine (K) change at amino acid 59. The base change at 466 results in analanine (A) to threonine (T) change at amino acid 156. The base changeat 870 does not result in an amino acid change at amino acid 290.

EXAMPLE 11

This example describes the cloning and sequencing of equine influenzaNeuraminidase (NA) protein nucleic acid molecules for cold-adaptedequine influenza viruses.

A. Nucleic acid molecules encoding cold-adapted equine influenza virusNA proteins were produced as follows. A PCR product was produced by PCRamplification using equine influenza virus DNA and primers M13 reverseprimer (REV) and T7 primer (T7), designated SEQ ID NO:1 and SEQ ID NO:2,respectively. A nucleic acid molecule of 1478 molecules encoding thecold-adapted NA protein, denoted nei_(ca)NA₁₄₇₈ with a coding strandhaving a nucleic acid sequence designated SEQ ID NO:87 was produced.

Translation of SEQ ID NO:87 indicates that nucleic acid moleculenei_(ca)NA₁₄₇₈ encodes a full-length protein of 470 amino acids,referred to herein as Pei_(ca)NA₄₇₀, having amino acid sequence SEQ IDNO:88, assuming an open reading frame in which the initiation codonsspans from nucleotide 29 through 31 of SEQ ID NO:87 and the terminationcodon spans from nucleotide 1439 through 1441 of SEQ ID NO:87. Theregion encoding Pei_(ca)NA₄₇₀, designated nei_(ca)NA₁₄₁₂, and having acoding strand comprising nucleotide 29 to 1441 of SEQ ID NO:87, isrepresented by SEQ ID NO:89.

EXAMPLE 12

This example describes the cloning and sequencing of the N-terminal andC-terminal regions of the equine influenza nucleoprotein (NP) nucleicacid molecules for wild type and cold adapted equine influenza virus.

A. Nucleic acid molecules encoding wild type and cold adapted equineinfluenza virus NP proteins were produced as follows. Wild-type strains1 and 2 were cloned into pCR2.1 (Invitrogen), and cold-adapted strains 1and 2 were cloned into pCR-Blunt cloning vector (Invitrogen). Allstrains were sequenced using M13 REV and T7 primers, SEQ ID NO: 1 andSEQ ID NO: 2, respectively.

B. N-Terminal Regions

A nucleic acid molecule of 738 nucleotides encoding the N-terminus ofthe wild-type strain 1 NP protein, denoted nei_(wt1) NP-N₇₃₈, with acoding strand having a nucleic acid sequence designated SEQ ID NO:104,was produced. Translation of SEQ ID NO:104 indicates that nucleic acidmolecule nei_(wt1) NP-N₇₃₈ encodes the N-terminal 246 amino acids of NPprotein, designated Pei_(wt1) NP-N₂₄₆, having amino acid sequence SEQ IDNO:105.

A nucleic acid molecule of 693 nucleotides encoding the N-terminus ofthe wild-type strain 1 NP protein, denoted nei_(wt1) NP-N₆₉₃, with acoding strand having a nucleic acid sequence designated SEQ ID NO:106was produced. Translation of SEQ ID NO:106 indicates that nucleic acidmolecule nei_(wt1) NP-N₆₉₃ encodes the N-terminal 231 amino acids of NPprotein, designated Peiw_(t1) NP-N₂₃₁, having amino acid sequence SEQ IDNO:107.

A nucleic acid molecule of 738 nucleotides encoding the N-terminus ofthe wild-type strain 2 NP protein, denoted nei_(wt2) NP-N₇₃₈, with acoding strand having a nucleic acid sequence designated SEQ ID NO:108,was produced. Translation of SEQ ID NO:108 indicates that nucleic acidmolecule nei_(wt2) NP-N₇₃₈ encodes the N-terminal 246 amino acids of NPprotein, designated Pei_(wt2) NP-N₂₄₆, having amino acid sequence SEQ IDNO:109.

A nucleic acid molecule of 693 nucleotides encoding the N-terminus ofthe wild-type strain 2 NP protein, denoted nei_(wt2) NP-N₆₉₃, with acoding strand having a nucleic acid sequence designated SEQ ID NO:110,was produced. Translation of SEQ ID NO:110 indicates that nucleic acidmolecule nei_(wt2) NP-N₆₉₃ encodes the N-terminal 231 amino acids of NPprotein, designated Pei_(wt2) NP-N₂₃₁, having amino acid sequence SEQ IDNO:111.

An N-terminal protein fragment of 245 amino acids of NP protein,designated Pei_(ca1) NP-N₂₄₅, having amino acid sequence SEQ ID NO:112,was generated.

A nucleic acid molecule of 690 nucleotides encoding the N-terminus ofthe cold-adapted strain 1 NP protein, denoted nei_(ca1) NP-N₆₉₀, with acoding strand having a nucleic acid sequence designated SEQ ID NO:113,was produced. Translation of SEQ ID NO:113 indicates that nucleic acidmolecule nei_(ca1) NP-N₆₉₀ encodes the N-terminal 230 amino acids of NPprotein, designated Pei_(ca1) NP-N₂₃₀, having amino acid sequence SEQ IDNO:114.

A nucleic acid molecule of 735 nucleotides encoding the N-terminus ofthe cold-adapted strain 2 Np protein, denoted nei_(ca2) NP-N₇₃₅, with acoding strand having a nucleic acid sequence designated SEQ ID NO:115was produced. Translation of SEQ ID NO:115 indicates that nucleic acidmolecule nei_(ca2) NP-N₇₃₅ encodes the N-terminal 245 amino acids of NPprotein, designated Pei_(ca2) NP-N₂₄₅, having amino acid sequence SEQ IDNO:116.

A nucleic acid molecule of 690 nucleotides encoding the N-terminus ofthe cold-adapted strain 2, denoted nei_(ca2) NP-N₆₉₀, with a codingstrand having a nucleic acid sequence designated SEQ ID NO:117, wasproduced. Translation of SEQ ID NO:117 indicates that nucleic acidmolecule nei_(ca2) NP-N₆₉₀ encodes the N-terminal 230 amino acids of NPprotein, designated Pei_(ca2) NP-N₂₃₀, having amino acid sequence SEQ IDNO:118.

C. C-Terminal Regions

A nucleic acid molecule of 679 nucleotides encoding the C-terminal ofthe wild type strain 1, denoted nei_(wt1) NP-C₆₇₉, with a coding strandhaving a nucleic acid sequence designated SEQ ID NO:119, was produced.Translation of SEQ ID NO:119 indicates that nucleic acid moleculenei_(wt1) NP-C₆₇₉ encodes the C-terminal 226 amino acids of NP protein,designated Pei_(wt1) NP-C₂₂₆, having amino acid sequence SEQ ID NO:120.

A nucleic acid molecule of 656 nucleotides encoding the C-terminal ofthe wild type strain 1, denoted nei_(wt1) NP-C₆₅₆, with a coding strandhaving a nucleic acid sequence designated SEQ ID NO:121, was produced.Translation of SEQ ID NO:121 indicates that nucleic acid moleculenei_(wt1) NP-C₆₅₆ encodes the C-terminal 218 amino acids of NP protein,designated Pei_(wt1) NP-C₂₁₈, having amino acid sequence SEQ ID NO:122.

A nucleic acid molecule of 679 nucleotides encoding the C-terminal ofthe wild type strain 1, denoted nei_(wt2) NP-C₆₇₉, with a coding strandhaving a nucleic acid sequence designated SEQ ID NO:123, was produced.Translation of SEQ ID NO:123 indicates that nucleic acid moleculenei_(wt2) NP-C₆₇₉ encodes the C-terminal 226 amino acids of NP protein,designated Pei_(wt2) NP-C₂₂₆, having amino acid sequence SEQ ID NO:124.

A nucleic acid molecule of 656 nucleotides encoding the C-terminal ofthe wild type strain 2, denoted nei_(wt2) NP-C₆₅₆, with a coding strandhaving a nucleic acid sequence designated SEQ ID NO:125, was produced.Translation of SEQ ID NO:125 indicates that nucleic acid moleculenei_(wt2) NP-C₆₅₆ encodes the C-terminal 218 amino acids of NP protein,designated Pei_(wt2) NP-C₂₁₈, having amino acid sequence SEQ ID NO:126.

A nucleic acid molecule of 656 nucleotides encoding the C-terminal ofthe cold-adapted strain 1, denoted nei_(ca1) NP-C₆₆₅, with a codingstrand having a nucleic acid sequence designated SEQ ID NO:127, wasproduced. Translation of SEQ ID NO:127 indicates that nucleic acidmolecule nei_(ca1) NP-C₆₆₅ encodes the C-terminal 222 amino acids of NPprotein, designated Pei_(ca1) NP-C₂₂₂, having amino acid sequence SEQ IDNO:128.

A nucleic acid molecule of 642 nucleotides encoding the C-terminal ofthe cold-adapted strain 1, denoted nei_(ca1) NP-C₆₄₂, with a codingstrand having a nucleic acid sequence designated SEQ ID NO:129 wasproduced. Translation of SEQ ID NO:129 indicates that nucleic acidmolecule nei_(ca1) NP-C₆₄₂ encodes the C-terminal 214 amino acids of NPprotein, designated Pei_(ca1) NP-C₂₁₄, having amino acid sequence SEQ IDNO:130.

D. Comparisons

Consensus sequences for the 5′ and 3′ ends of the NP gene weregenerated, but there is an approximately 148 base pair gap in the datathat prevents the formation of a contiguous sequence when compared toprotein and DNA data currently in the GenBank databases (a nucleoproteinfrom GenBank named flanpg; Gorman et al, J. Virol. 64, 1487 (1990)). Theflanpg sequences were used to compare the 5′ and 3′ fragments of thepresent NP genes to get relative positions that are used to designateposition for the various differences in the bases between the Wild Typeand the ca strains. The flanpg sequence allows for the approximately 148base pairs that are missing between the 5′ and 3′ fragments needed toconstruct the entire gene.

The DNA codes for the N-terminal 735–738 bases and the C-terminal665–679 bases with a gap of approximately 149–166 bases between the twofragments of the Wild Type and cold-adapted (ca) strains, respectively.There are seven discrepancies between the wild type and ca strains. Theca strains have discrepancies at base 146 (G to T) and at base 228 (A toG). At base 492, the wt1 only has a discrepancy versus the other strainsof (A to C). At base 541, the ca1 only has a discrepancy versus theother strains of (C to A). There is a base discrepancy at position 645in the DNA (G to A). At base 670, the ca strains have a discrepancy (Gto A). At base 1019, the wt1 only has a discrepancy versus the otherstrains of (G to A). There are no other points of discrepancy betweenthe full-length Wild Type and ca strains.

The full-length sequence is proposed to consist of 1497 bases, howeverthere is no overlap between the 5′ and 3′ ends of the DNA sequencesgenerated. The sequences listed are for the 5′ end and the 3′ end of theopen reading frame of the gene. The gene is missing approximately 148base pairs between these two fragment sequences that would generate thecomplete gene sequence.

A comparison of all the Wild Type and ca strain clones (wt1, wt2, ca1,and ca2) by amino acid translation for the protein sequence coding onlyfor the open reading frame (ORF) from the 5′ and 3′ DNA fragments wascompared to the GenBank nucleoprotein noted above. The DNA codes for theN-terminal 230 amino acids and the C-terminal 214 amino acids with a gapof approximately 55 amino acids between the two fragments. There areseven discrepancies between the ca and Wild Type strains. The ca strainshave a discrepancy (G to V) at amino acid 34, and at amino acid 61 (I toM). At amino acid 149, the wt1 only has a discrepancy versus the otherstrains of (Q to H). At amino acid 166, ca1 only has a discrepancyversus the other strains of (L to M). Although there is a basediscrepancy at position 600 in the DNA sequence, there is no change inthe corresponding amino acid sequence. At amino acid 209, the ca strainshave a discrepancy (G to S). At amino acid 325, the wt1 only has adiscrepancy versus the other strains of R to K). There are no otherpoints of discrepancy between the fill-length Wild Type and ca strains.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims.

1. An isolated equine influenza protein that comprises the amino acidsequence of SEQ ID NO:28.
 2. A composition comprising the isolatedequine influenza protein of claim 1 and an excipient.